Our research focuses on complementary and alternative medicines (CAM) that possess anti- cancer properties. In the proposed study, we will analyze the mechanism of action of Withaferin-A (WA), a CAM based herbal used extensively in African and Asian countries for the treatment of various ailments, including cancer. Our preliminary in vitro data suggest WA targets colon cancer cells by down-regulating cleaved NOTCH1, pAkt, pS6K, p4E-BP1, and Bcl- 2, and simultaneously promoting caspase-3 activation and PARP cleavage. Based on these results, we hypothesize WA will effectively inhibit colon cancer growth due to its ability to inactivate Notch-1/Akt/mTOR-mediated pro-survival signaling, and induce apoptosis in colon cancer cells. To address our hypothesis, we propose: study the mechanism(s) of WA-mediated Notch-1/Akt/mTOR inhibition in colon cancer (Aim 1), and determine the in vivo efficacy of WA and its chemosensitization effect on colon cancer xenografts that over-express either Akt or mTOR (Aim 2). For our in vitro studies, we will use RT-PCR, Western Blotting, SiRNA strategies, immunoprecipitation, cell viability assays, apoptotic assays, kinase assays, transient transfection, gene transcription assays, pharmacological blocking, and immunocytochemistry to clarify the impact of WA on the Notch-1/Akt/mTOR signaling axis. For our in vivo studies, colon cancer cells will be stably transfected with either constitutively active Akt or mTOR. Using these transfectants, xenografts will be formed in nude mice, and the effect of WA on the tumor bearing animal models will be studied. Additionally, to determine the chemosensitization effect of WA on tumor bearing animal models, we will treat mice with WA and a sub-lethal dose of 5-Fluorouracil (5-FU). Necroscopy, histopathology, and immunohistochemical (Notch-1, Akt, mTOR, cyclin D1, Par-4 and Bid) analyses will be performed on the tumor sections following the termination of the study. Additionally, HPLC will be performed to determine serum concentrations of WA in the nude mice models. Our long term goal is to encourage the use of CAMs in a clinical environment, where these agents can be best used for their chemopreventive and chemotherapeutic properties. Our preliminary data indicate WA is one such compound, and is a viable candidate for investigating its clinical potency against colon cancer cells. PUBLIC HEALTH RELEVANCE: Colorectal cancer is the third most frequently diagnosed cancer in men and women in the United States, and, although its mortality rates have decreased due to early detection and adjuvant therapies, the disease is incurable once metastases develop. Complementary and alternative systems of medicine (CAM) may offer novel and effective therapies not yet explored by conventional medicine. The proposed study of the dietary supplement Withaferin-A (WA), the major bioactive compound from Withania somnifera), will provide molecular level insight into the mechanism of action of Withaferin-A against colon cancer, and clarify the clinical potential of WA with respect to the chemoprevention and/ or chemotherapy of colon cancer.